Abstract

The aim of the study was to evaluate frequency, types and renal manifestations in patients with chronic hepatitis C. Our research included 109 patients with chronic hepatitis C. They were examined and treated at the Department of Infectious Diseases of Bogomolets National Medical University and SI "Institute of Nephrology, NAMS of Ukraine." Renal manifestations of chronic hepatitis C were diagnosed in 12.8% of cases. Main clinical and laboratory manifestations included general weakness (was detected in 14 (100%) patients), arterial hypertension (was observed in 11 (78.6%) patients), edema (was observed in 9 (64.3%) patients), proteinuria (was observed in 14 (100%) patients) and microhematuria (was observed in 11 (78.6%) patients), nephrotic syndrome was observed in 9 (64.3%) patients. Arthralgia was observed in 3 (21.4%) patients, and purpura in 1 (7.1%) patient, eGFR reducing was in 9 (64.3%) patients. In 8 (66.7%) patients of our study elevated levels of serum cryoglobulins (from mild to high levels) were detected. According to our data long-term HCV-infection and cirrhosis were considered risk factors for the development of extrahepatic immunological diseases, including renal disease. An important role of renal biopsy in the differential diagnosis and determination of further treatment plan of patients with renal impairment in chronic hepatitis C was pointed out. We noted that kidney damage in patients with chronic hepatitis C in most of the cases were associated with the cryoglobulinemic syndrome. We performed renal biopsies in 12 patients with symptoms of chronic kidney disease and cryoglobulinemic glomerulonephritis was detected in 7 (58.3%) patients. Other types of kidney damage detected in our study included membranoproliferative glomerulonephritis without cryoglobulinemia, membranous nephropathy, focal segmental glomerulosclerosis, mesangioproliferative glomerulonephritis. Keywords: Chronic hepatitis C; Cryoglobulinemic syndrome; Membranoproliferative glomerulonephritis

Introduction

Chronic hepatitis C (CHC) is considered as a systemic disease,
with damage of not only the liver, but also with a variety of extrahepatic manifestations. The leading place among them
belongs to mixed cryoglobulinemia. Since the discovery of the
hepatitis C virus (HCV) in 1988 it has been identified as the main
cause of mixed cryoglobulinemia, as it was found in 90% of
patients with cryoglobulinemia [1,2]. The classic manifestations
of Cryoglobulinemic Syndrome (CGS) is weakness, arthralgia
and purpura (Meltzer triad). Other symptoms include damage
of other systems such as kidneys, peripheral (less frequently central) nervous system, gastrointestinal tract, lungs, etc. [3,4].
Renal disease in patients with HCV– infection is considered to be
a proven fact [5,6]. The most common is Membranoproliferative
Glomerulonephritis (MbPGN), which is associated with the II type
of cryoglobulinemia (cryoglobulinemic Glomerulonephritis (GN).
Less common MbPGN without cryoglobulinemia, Membranous Glomerulonephritis (MGN), Focal Segmental Glomerulosclerosis
(FSGS), mesangioproliferative glomerulonephritis, fibrillary
glomerulonephritis and imunotaktoid glomerulopathy [7- 17]. There are also other atypical cases of kidneys’ damage
in HCV–infection–IgA nephropathy, rapidly progressive
glomerulonephritis, thrombotic microangiopathy [18-20].
Isolated cases of albuminuria without kidney damage in HCV
infection were also described [16]. In general, the prognosis
for HCV–associated glomerulonephritis remains poor, not only
because of the progression of kidneys’ disease and development
of liver cirrhosis, but also through the high risk of cardiovascular
complications

Methods

The study was conducted at the Department of Infectious Diseases of Bogomolets National Medical University and at
the Department of Nephrology and Dialysis of SI "Institute of
Nephrology of NAMS of Ukraine." We observed 109 patients with
CHC. Patient selection criteria was diagnosed chronichepatitis C, according to the classification of chronic hepatitis, proposed
at the International Congress of Gastroenterology (Los Angeles,
1994) [21] which was verified by the identification of specific
serological and molecular genetic markers of HCV. CGS was
diagnosed according to the recommendations of the European
Association for the Study of Liver (EASL, 2012) based on presence
in serum of mixed cryoglobulins (CGs), antibodies to HCV,
positive rheumatoid factor (RF), reduction of complement C4,
morphological (leukocytoclastic vasculitis, infiltration of tissues
by monoclonal B-cells) and clinical (purpura, fatigue, arthralgia,
MbPGN, peripheral neuropathy) criteria [22].

Clinical examination, general laboratory tests, biochemical tests,
serological and molecular genetic studies in dynamics were
performed in accredited commercial laboratories.

The cryoglobulins levels were detected using spectrophotometry
method by calculating the difference of the optical density of
the solution of serum in a buffer (pH=8.6) at 4°C and 37°C [4].
Glomerular filtration rate (GFR) was estimated according to
MDRD [23].

Morphological study of renal samples were performed in 12
patients. Presence of clinical and/or laboratory manifestations
of chronic kidney disease in patients with chronic hepatitis C
for the period of 3 months or longer were the main indications
for renal biopsy. Renal samples were obtained by percutaneous
needle biopsy of the kidney by a standard technique under the
supervision of the ultrasound scanner. We used the methods of
light, immunofluorescent and transmission electron microscopy.
For light microscopy paraffin sections were stained with
hematoxylin and eosin, Masson's trichrome, Congo red, Schiff
reagent. Jones silver staining was also used. Direct method of
Coons with FITC labeled polyclonal antibodies to IgA, IgG, IgM, and light chains κ and λ, fibrinogen and complement fractions
C1q and C3 in dilution 1:10 - 1:60 (DAKO, Denmark) was used
on frozen sections of kidneys for the immunofluorescence study.
Fluorescence microscope Olympus BX-51 (Japan) was used for
the light and immunofluorescence studies. The material for
the electron-microscopic study was embedded in EPON-araldit
mixture using an ultramicrotome LKB-III (Sweden) for production
of semithin and ultrathin sections. Semithin sections were stained
with toluidine blue. Ultrathin sections were contrasted with 2%
solution of uranil acetate and plumbum citrate and examined
with an electron microscope TEM-125K (SELMI, Ukraine) at an
accelerating voltage of 60 kV. Morphological diagnosis was based
on morphological classification of renal diseases for nephrology
practice, 2010 [23].

Statistical analysis of the results was performed by using
descriptive statistics. Calculations of the results were performed
using the software package "Statistica 6.0".

Results

We observed 109 patients with CHC. Renal lesions were
diagnosed in 14 (12.8%) patients. Baseline characteristics of the
patients presented in Table 1.

Results of the urinalyses showed that all patients had proteinuria
which ranged from 0.24 g/l to 9.5 g/l. Erythrocyturia (from
10 erythrocytes to solid in sight) was observed in 11 (78.6%)
patients. Leykocyturia was observed in 9 (64.3%), cylindruria – in
12 (85.7%) patients. Nephrotic syndrome occurred in 9 (64.3%)
patients.

Thus, according to the results of clinical and laboratory
examination the symptoms of kidney damage in patients with
CHC were dominant and the activity of hepatitis was minimal in
the majority of these patients. However, it should be emphasized
that hepatic fibrosis F3-F4 was detected in 5 (35.7%) patients. Renal needle biopsy with morphological study was performed in
12 patients with chronic kidney disease. In other two patients
with transient proteinuria were no indications for biopsy.
Cryoglobulinemic GN was diagnosed in 7 of 12 patients (58.3%)
according to the results of morphological studies of renal
specimens. Two patients had MbPGN without cryoglobulinemia.
Membranous nephropathy, FSGS and mesangioproliferative GN
was diagnosed in 1 patient respectively.

Increased levels of serum CGs (from mild to high levels) were
diagnosed in 8 (66.7%) patients. Seven of these patients had
cryoglobulinemic GN and one patient had FSGS.

An increased size of the glomeruli with global endocapillary
hypercellularity and excess of mesangial matrix was observed
in all cases of HCV-associated MbPGN with and without
cryoglobulinemia using light and electron microscopic study.
Diffuse thickening of the glomerular basement membrane (GBM)
was differently expressed in different capillary loops. Thickening
parts of GBM at the test samples stained with Schiff reagent and
Jones silver impregnation looked like double-contoure (Figure
1a). Infiltration by mononuclear cells of varying degrees of
severity were observed in 4 (33.3%) cases. Cellular and fibrouscellular
crescents were detected in 2 (16.7%) cases. Numerous
subendothelial and mesangial electron-dense deposits were
identified using electron-microscopic study. They were small
and discrete and also large and elongated. Additional membrane
at the pericapillary part was often seen as well as interposition
of mesangial cells. Subepithelial electron-dense deposits were
observed in 2 (16.7%) cases. The fusion of small processes of
podocytes was noted as a characteristic feature.

The focal tubular atrophy was detected in 6 (50.0%) patients
and accumulation of foamy macrophages was found in the
interstices of 3 (25.0%) patients. The areas of fibrosis were
detected in 5 (41.6%) patients, focal inflammatory infiltration - in
7 (58.3%) patients. 2 (16.7%) patients had features of myointimal
hyperplasia of small arteries.

A characteristic feature of cryoglobulinemic GN was the presence
of eosinophilic and PAS-positive hyaline thrombi in the lumen of
glomerular capillaries. They were either single (in some capillary
loops of the glomeruli) or numerous (in most of the glomerular
loops) (Figure 1b). The expressive glomerular mononuclear
cells infiltration was also found. Data obtained during electronmicroscopic
examination showed the presence of subendothelial,
mesangial and intraluminal electron-dense deposits. In some
cases it was possible to detect their microtubule or crystalloid
structure by high magnification.

Immunofluorescence investigation in all cases of HCV-associated
MbPGN showed small and large granullar deposits of C3, IgM and
IgG along the capillary walls and at the mesangium. The highest
intensity of luminescence was typical for C3 and IgM (Figures 1c-
1e). Intraluminal deposits in cases of cryoglobulinemic GN were
positive for IgM and IgG, as well as for C3. Immunofluorescence
investigation data of patients with HCV-associated MbPGN is
shown in Figure 2.

Figure 2: Average luminescence intensity of immunoglobulins and complement in patients with HCV– associated MbPGN, the results of
immunofluorescence studies, n=9.

Discussion

Renal disease can develop within a few years or even decades
after infection with HCV. According to the literature data, the most
common form of kidneys’ damage in CHC is cryoglobulinemic
MbPGN (PbPGN type I), which is usually associated with type
II of cryoglobulinemia [5-10]. Usually MbPGN is diagnosed in
patients with long-term HCV-infection. In our study the duration
of HCV-infection was 10.2 ± 3.7 years and the average age of the
patients was 40.3 ± 12.6 years. According to the literature data
MbPGN type I is often diagnosed in patients with HCV-associated
liver cirrhosis [24,25]. Hepatic fibrosis F3-F4 was diagnosed in 5
(35.7%) patients in of our research. Thus, long-term HCV-infection
and hepatic fibrosis F3-F4 are risk factors for the development of
kidney damage in patients with CHC.

Clinical course of the disease in patients with impaired renal
function usually include other symptoms of cryoglobulinemia:
purpura, arthralgia, fatigue, which were also seen in patients
of this study (general weakness – in 14 (100%) patients,
arthralgia - in 3 (21.4%) patients, purpura - in 1 (7.1%) patient.
According to the literature data, symptoms of kidney damage
approximately occur in one third of patients with CGS. Renal
symptoms of cryoglobulinemia include nephrotic syndrome,
isolated proteinuria (that can be the only symptom of kidney
damage in CHC for a long time), microscopic hematuria, 80% of
patients develop hypertension [26]. In our study, hypertension
was diagnosed in 11 (78.6%) patients, swelling of the lower
extremities and face - in 9 (64.3%) patients, proteinuria was
found in all patients, microhematuria–in 11 (78.6%) patients.
Nephrotic syndrome occurred in 9 (64.3%) patients.

The results of this study also confirm studies of foreign authors:
the majority of patients–7 (58.3%)–with chronic hepatitis
C and kidney lesions were diagnosed morphologically with
cryoglobulinemic GN, the major pathogenetic link of which is the
deposition of the CG in the capillaries and glomerular mesangial.
However, there were other types of renal injury in this study–
MbPGN without cryoglobulinemia (in case of which the
deposition of immune complexes (HCV antigens, IG, complement
fragments) occurred in the mesangium); MGN (in case of which
the subepithelial deposition of immune complexes (antigens of
HCV, IG, complement fragments) develops); FSGS (associated
with direct damage of podocytes caused by hepatitis C virus)
and mesangioproliferative GN (caused by the direct action of the
hepatitis C virus on the mesangium through TLR-3 or MMP-2)
[27,28].

Conclusions

Thus, our study showed that the renal manifestations of HCV
infection appear in 12.8% of cases. The main clinical and
laboratory manifestations in patients of our study were general
weakness, hypertension, lower extremities and face swelling,
proteinuria and microhematuria, nephrotic syndrome and eGFR
reduction. We also observed such symptoms as arthralgia and
purpura. In our study levels of serum CGs were elevated in 8
(66.7%) patients (from mild to high levels).

The results of our study showed that the long-term HCV-infection
and cirrhosis were risk factors for the development of renal
lesions.

Needle kidney biopsy is important in the differential diagnosis,
since clinical manifestations of kidney damage are usually nonspecific. According to the results of our study, the majority
of renal lesions in chronic hepatitis C was linked to the CGS,
as cryoglobulinemic GN was found in 7 (58.3%) of 12 patients.
There were also other types of kidney damage – a membranous
nephropathy, MbPGN without cryoglobulinemia, focal segmental
glomerulosclerosis and mesangioproliferative GN.